The present invention relates to a novel pharmacophore model for identifying compounds that are useful for the inhibition, alteration or prevention of the binding of the integrin VLA-4 to its ligands. This invention also relates to methods of discovering molecules which may inhibit VLA-4 binding to its ligands as well as novel molecules which have features which map to the claimed models.
In recent years, rational drug design has become a common approach to identifying new drugs in the pharmaceutical industry. This approach requires selecting a protein target molecule which plays a critical role in a physiologically relevant biological pathway. The chemist typically begins with the natural ligand as the lead and modifies it to produce a compound with the desired properties. The natural ligand or substrate of this protein is manipulated to produce an enzyme inhibitor, or an agonist or antagonist for a receptor, depending upon the identified therapeutic need, capitalizing upon knowledge of what is known about the mechanism of action of the protein-ligand complex.
Most cell receptors have a developed pharmacology of agents that act as agonists or antagonists. However, despite extensive pharmacological research and the development of many new methodologies and laboratory techniques, certain receptors, and/or their action still remain elusive and no desirable antagonists have yet been discovered to inhibit or modulate their activity.
Additionally, often certain agonists or antagonists of a particular cell receptor are known, however, there remains a need for methods of identifying new inhibitors, new molecular entities and methods to quickly and effectively determine whether a particular compound possesses a desired pharmacological activity.
Cell adhesion is one of the fundamental mechanisms underlying numerous biological phenomena, such as, for example, the adhesion of hematopoietic cells to endothelial cells, and the subsequent migration of those hematopoietic cells out of the blood vessels and to the site of injury. Thus, cell adhesion is known to play a role in numerous pathologies such as inflammation and immune reactions.
xcex14xcex21 integrin, also known as very late antigen-4 (xe2x80x9cVLA-4xe2x80x9d), is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell-matrix adhesive interactions. It serves as a receptor for the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-1 (xe2x80x9cVCAM-1xe2x80x9d), as well as to the extracellular matrix protein fibronectin. Results of several in vivo experiments suggest that the inhibition of VLA-4 dependent cell adhesion may prevent, inhibit or alter several inflammatory and autoimmune pathologies.
In order to identify the minimum active amino acid sequence necessary to bind VL-4, Komriya et al. snthesized a variety of overlapping peptides based on the amino acid sequence of the CS-region (the VLA-4 binding domain) of a particular species of fibronectin. (xe2x80x9cThe Minimal Essential Sequence for a Major Cell Type-Specifice Adhesion Site (CS1) Within the Alternatively Spilced Type III Conencting Segment Domain of Fibronectin Is Leucine-Aspartic Acid-Valinexe2x80x9d, J. Biol. Chem., 266 (23), pp. 15075-79 (1991). They identified an 8-amino acid peptide, SEQ ID NO:1 Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two smaller overlapping pentapeptides, SEQ ID NO:2 Glu-Ile-Leu-Asp-Val and SEQ ID NO:3 Leu-Asp-ValPro-Ser, that possessed inhibitory activity against FN-dependent cell adhersion. These results suggested that the tripeptide Leu-Asp-Val was the minimum sequence for cell-adhesion activity. It was later shown that Leu-Asp-Val binds only to lymphocytes that express an activated form of VLA-4, thus casting doubt on the utility of such a peptide in vito. (E. A Wayner et al., xe2x80x9cActivationxe2x80x94Dependent Recognition by Hematopoietic Cells of the LDV Sequence in the V Region of Fibronectinxe2x80x9d, J. cell. Biol., 116(2), pp. 489-497 (1992)). However, certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo (T. A. Ferguson et al., xe2x80x9cTwo Intergrin Binding Peptides Abrogate T-Cell-Mediated Immune Responses in Vivoxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991); and S. M. Wahl et al., xe2x80x9cSynthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitmentxe2x80x9d, J. Clin. Invest., 94, pp. 655-62 (1994)).
A cyclic pentapeptide, SEQ ID NO:4 Arg-Cys-Asp-Tpro-Cys (wherein Tpro denotes 4-thioprline), which can inhibit both VLA-4 and VLA-5 adhesion to FN has also been described. (See, e.g., D. M Nowlin et al. xe2x80x9cA Novel Cyclic Pentapeptide Inhibits xcex14xcex21 and xcex15xcex21 Integrin-mediated Cell-Adhesionxe2x80x9d, J. Biol. Chem., 268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862. This pentapeptide was based on the tripeptide sequence Arg-Gly-asp from FN which had been know as a common motif in the recognition site for several extracellular-matrix proteins.
Examples of other VLA-4 inhibitors have been reported, for example, in copending United States patent application U.S. Ser. No. 08/376,372, specifically incorporated by reference herein. U.S. Ser. No. 376,372 describes linear peptidyl compounds containing xcex2-amino acids which have cell adhesion inhibitory activity. International patent applications WO 94/15958 and WO 92/00995, specifically incorporated by reference, describe cyclic peptide and peptidomimetic compounds with cell adhesion modulating activity. International patent applications WO 93/08823 and WO 92/08464 (specifically incorporated by reference herein) describe guanidinyl-, urea- and thiourea-containing cell adhesion modulating compounds. U.S. Pat. No. 5,260,277 describes guanidinyl cell adhesion modulation compounds, and is also specifically incorporated herein.
As discussed above, it is desirable for several reasons to approach the discovery of new drugs in a rational as opposed to a random manner. Thus, rather than making random modifications to a compound, one can rationally optimize the compound.
Ideally, a three dimensional model of the binding mode of inhibitors to a receptor is sought such that a correlation between the structure of the compound and its effect on biological activity can be derived. Several general approaches exist for determining the three dimensional quantitative structure activity relationships of compounds and their receptors or ligands, including, but not limited to: CATALYST(trademark) (Greene et al., 1994, xe2x80x9cChemical Function queries for Three dimensional database searchxe2x80x9d, J. Chem. Inf. Comp. Sci., 34, 1297-1308), DISCO (Martin Y. C., et al., 1993, xe2x80x9cA Fast new approach to pharmacophore mapping and its application to dopinergic and benzodiazepine agonistsxe2x80x9d, J. Comp. Aided Mol. Design, 7, 83-102), COMFA (Cramer R. D., 1988, xe2x80x9cComparative molecular field analysis [COMFA] 1. Effect of Shape on Binding of Steroids to Carrier Proteinsxe2x80x9d, J. Am. Chem. Soc., 110, 5959-5967), Apex3D (Golender, V. E. And Vorpagel, E. R., 1993, xe2x80x9cComputer-assisted pharmacophore identificationxe2x80x9d, Three dimensional-QSAR in Drug Design:Theory, Methods and Applications, ESCOM Science Publ., Netherlands). Once a three dimensional model is built it can be useful in identifying novel compounds. For example, Kiyama et al. were able to identify novel AII antagonists based upon a three dimensional model of known AII inhibitors. (1995, xe2x80x9cNovel AII receptor antagonists. Design, synthesis, and in-vitro evaluation of dibenzo[a,d] cycloheptene and dibenzo[b,f] oxepin derivatives. Searching for bioisoteres of biphenyltetrazole using a Three dimensional search techniquexe2x80x9d, J. Med. Chem., 38, 2728-2741).
In general, there are several fundamental forces which govern the molecular recognition between a drug and its receptor, including, for example, hydrogen-bonding forces, electrostatic and hydrophobic interactions. Until recently most descriptions of inhibitors have been based upon two dimensional atomic topology diagrams which describe chemical structures (e.g. indole ring, carbonyl oxygen). Although these diagrams may be useful, they are somewhat limited in the information that they provide regarding the details of the biological activity of compounds. The availability of additional information would aid chemists in identifying novel compounds with a particular biological activity relatively quickly, cheaply and with a relatively high level of success.
An alternative to the two dimensional atomic topology approach (Greene et al., 1994, xe2x80x9cChemical Function queries for Three dimensional database searchxe2x80x9d, J. Chem. Inf. Comp. Sci., 34, 1297-1308) describes compounds on the basis of chemical features which take into account the type of binding interaction of the chemical substructure. (FIG. 1; e.g. H-bonding donor, hydrophobe). One advantage of this approach is that it allows for a more general description of compounds, and accounts for its possible interactions with a receptor. The recognition that alternative chemical structures can present the same chemical features is central to drug discovery.
Examples of the use of the feature-based description of compounds to describe potent antagonists which differ in chemical structure but are similar in the chemical features they present exist. These include, for example, angiotensin converting enzyme antagonists (Sprague, 1994, xe2x80x9cBuilding a hypothesis for Angiotensin Converting Enzyme Inhibitionxe2x80x9d, MSI Inc., 16 New England Executive Park, Burlington, Mass. 01803) and A2 antagonists (Sprague, 1994, xe2x80x9cBuilding a hypothesis for AII Antagonismxe2x80x9d, MSI Inc., 16 New England Executive Park, Burlington, Mass. 01803).
Despite these advances, there remains a need for a model of a VLA-4 inhibitor which can be used to identify new specific inhibitors of cell adhesion, particularly for methods of identifying novel, specific inhibitors of VLA-4 cell adhesion. The availability of additional information would aid those skilled in the art to identify novel compounds with a particular biological activity quickly, inexpensively, and with a relatively high level of success. Ideally, such methods would allow practitioners to predict the inhibitory activity of novel compounds which would provide useful agents for treatment, alteration, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 binding.
Accordingly, the present invention is directed to a model of a VLA-4 inhibitor, methods of identifying new inhibitors, and new compounds which inhibit VLA-4 activity which map to the model, which substantially obviate one or more of the problems due to the limitations and disadvantages of the related art.
To achieve the features and advantages of the invention, as embodied and broadly described herein, the present invention relates to a three dimensional pharmacophore model of a compound having VLA-4 inhibitory activity. The claimed model 1 comprises certain features defined by the following tolerance and three dimensional coordinates x, y and z. Specifically the model comprises NEG (xe2x80x9cNxe2x80x9d)
and at least three features selected from the group consisting of
The coordinates of the claimed models define the relative relationship between the features, and therefore those skilled in the art will readily recognize that the specific coordinates are dependent upon the particular coordinate system used, and thus, although rotation or translation of these coordinates may change the specific values of the coordinates, the coordinates will, in fact, define the claimed models.
Those skilled in the art should recognize that the claimed models are not without standard error. Thus, the claimed models are intended to encompass any model comprising the identified features and having a root mean square of equivalent features of less than about 2 xc3x85.
More specifically, the model 1 of the invention comprises the negative ionizable feature xe2x80x9cNEGxe2x80x9d, and at least four features selected from the group consisting of features 1-7. In other embodiments, the model may encompass 5-7 of the features, in addition to NEG.
In other embodiments, the applicant""s invention relates to compounds which xe2x80x9cmapxe2x80x9d to the claimed model. As used herein, the terms xe2x80x9cmapxe2x80x9d and xe2x80x9cfitxe2x80x9d are used interchangeably to denote the correspondence between some or all of the features in a hypothesis and the chemical substructure of a particular conformer of a compound that satisfy those features, as computed by xe2x80x9ccatalystxe2x80x9d (xe2x80x9cHypothesis in Catalyst,xe2x80x9d MSI Inc., New England Executive Park, Burlington, Mass. 01803; Greene, I., 1994, J. Chem. Inf. Sci., xe2x80x9cChemical Function Queries for 3D Database Search,xe2x80x9d 34, 1297-1308). In additional embodiments, compounds having an IC50 value in a VLA-4 direct binding assay in the range of from about 100 xcexcm to about 1 xcexcm, and which comprise features which map to NEG, and an additional 3-7 features of the model, are encompassed.
In yet other embodiments, applicants have discovered novel methods for identifying chemical compounds having an IC50 value in a VLA-4 direct binding assay in the range of from about 100 xcexcM to about 1 xcexcM. The methods of the invention generally encompass selecting an experimental compound structure to be evaluated for VLA-4 inhibitory activity. The three dimensional structure of said compound is then obtained, and the structure of the experimental compound is then superimposed upon the VLA-4 model of the invention and evaluated to determine if the experimental compound xe2x80x9cfitsxe2x80x9d the model. If the experimental compound fits the model, it is then tested in a direct binding assay to determine whether or not said experimental compound has the desired inhibitory activity. The compounds of the invention preferably have an inhibitory activity in the range of about 100 xcexcM to about 0.5 nM, preferably of less than about 50 xcexcm, more preferably less than about 500 nM, and most preferably, less than about 50 nM.
In yet other embodiments, the claimed invention relates to model 2, a three dimensional pharmacophore model of a compound having VLA-4 inhibitory activity. Model 2 comprises Neg (xe2x80x9cNxe2x80x9d) as defined below, and at least four of features 1 through 8.
Preferably, the model comprises at least 5 to 8 of the features 1-8 of Model 2. Additionally, applicants invention relates to compounds which fit Model 2 and have features which map to between 4 and 8 of the features of Model 2. The compounds of the invention preferably have an inhibitory activity in the range of about 100 xcexcM to about 50 nM, preferably of less than about 50 xcexcm, more preferably less than about 500 nM, and most preferably, less than about 50 nM.
In other embodiments, the claimed invention relates to methods for identifying chemical compounds having VLA-4 inhibitory activity using Model 2 in a manner similar to that described above for Model 1, as well as to compounds obtained by the claimed methods.
In still other embodiments, the claimed invention relates to a third three dimensional pharmacophore model of a compound having VLA-4 inhibitory activity. Model 3 comprises the following features:
As discussed above in relation to Models 1 and 2, the invention also encompasses methods for identifying desired compounds using Model 3, as well as novel compounds which map to Model 3. Preferably, the novel compounds encompassed by the claims have the preferred IC50 values discussed above.